Phospholipase enzymes catalyze the removal of fatty acid residues from phosphoglycerides. Specifically, phospholipase A2 (PLA2) cleaves the ester bond at the 2 position of the glycerol moiety of membrane phospholipids giving rise to equimolar amounts of arachidonic acid and lysophospholipids. Although PLA2 preferentially cleaves arachidonic acid from phospholipids, arachidonic acid is generated secondarily from intermediates of the S1, phospholipase C- and phospholipase D-activated pathways.
Although the known PLA2s were originally divided into groups by source organism, shown in parentheses, and their primary amino acid sequences, they are now characterized by a growing list of other attributes. Group 1 includes the 80-90 kD PLA2s (mammalian) which are active at pH 6-8 and dependent on the presence of calcium ion (Ca.sup.++). Group II is a mixture of approximately 14 kD, secreted PLA2s (snake) which bind heparin and are inhibited by dexamethadone, dithiothreitol and deoxycholate. Group III is the cytosolic, 100 kD PLA2s (honey bee venom). The prokaryotic versions of PLA2 are produced by bacteria such as Streptomyces violaceoruber.
Several recent scientific studies reveal pertinent facts towards the characterization of PLA2s and their lipolytic activity. Van den Berg B et al. (1995; EMBO J 14:4123-31) reported that PLA2 appears to be more active in the degradation of high molecular weight aggregates than of monomers. In vitro experiments by de Carvalho M G et al. (1995; J Biol Chem 270:20439-46) showed that with a bilayer substrate, PLA2 preferentially and sequentially deacylates sn-2 and then sn-1 acyl groups. Ross et al. (1995; J Neurochem 64:2213-21) reported that in the temporal cortex of the human brain, PLA2 activity was higher in membrane fraction than in the cytosolic fraction.
Arachidonic acid, the product of PLA2 activity, is processed into bioactive lipid mediators such as lyso-platelet-activating factor (lyso-PAF) or shuttled into pathways for the synthesis of eicosanoids. In fact the release of arachidonic acid from membrane phospholipids is the rate-limiting step in the biosynthesis of the four major classes of eicosanoids (prostaglandins, prostacyclins, thrombosanes and leukotrienes) involved in pain, fever, and inflammation. Furthermore, leukotriene-B4 (LKB4) is known to function in a feedback loop which induces further increased PLA2 activity.
PLA2 has many other known activators which include tumor necrosis factor (Jaattela M et al. (1995) Oncogene 10:2297-305); the protein phosphatase inhibitor, okadaic acid (Gewert K and R Sundler (1995) Biochem J 307:499-504); the neuroleptics, fluphenazine and thioridazine (Trzeciak H I et al (1995) Eur Arch Psychiatry Clin Neurosci 245:179-182); the mammalian phospholipase A2-activating protein (PLAP; Yamada H and Bitar KN (1995) Biochem Biophys Res Commun 217:203-10); and the eicosanoids, LKB4, 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (Wijkander J et al (1995) J Biol Chem 270:26543-9). Epidermal growth factor specifically induces serine phosphorylation-dependent and calcium-dependent activation of cytosolic PLA2 (Schalkwijk C G et al. (1995) Eur J Biochem 231:593-601).
Inhibitors of PLA2 are useful in the regulation of the signaling cascades that result from or correlate with PLA2 activity. One pronounced example of this signaling is seen in sepsis where the increase in PLA2 was found to be more than 12,000-fold normal and PLA2 was associated with complement C3-derived anaphylatoxin. PLA2 inhibitors include chemical molecules such as p-bromophenacyl bromide and biological molecules such as the specific inhibitor, thielocin A1 beta, produced by a fungus (Tanaka et al. (1995) Eur J Pharmacol 279:143-8) and nonspecific inhibitors such as glucocorticoids.
Fortes-Dias C L et al. (1994; J Biol Chem 269:15646-51) have isolated and characterized a PLA2 inhibitor from the plasma of a South American rattlesnake, Crotalus durissus terrificus. This 20-24 kD protein, designated Crotalus neutralizing factor (CNF), appears to self-associate as a 6-8 oligomeric aggregate. The crotoxin molecule which CNF neutralizes is active only as a dimer and consists of an acidic molecule (CA) associated with one of two basic isoforms of PLA2 (CB.sub.1 and CB.sub.2). CNF actually displaces CA to form a stable association with one of the CB molecules. This displacement inactivates the neurotoxic, cardiotoxic, myotoxic, anticoagulent and platelet-activating activities of crotoxin.
The full length 840 bp cDNA of CNF was cloned from Crotalus liver tissue. The nucleotide sequence encodes a 19 residue signal peptide and a 181 residue mature protein with 16 cysteines, a pl of 5.45, and a possible glycosylation site at N.sub.157. Fortes-Dias states that the cDNA contains noncoding sequence and lacks a putative polyadenylation site. In inhibitory assays, the acidic CNF molecule also inhibits the activity of bee venom, and in 100-fold excess in plasma, porcine pancreatic PLA2.